Eph receptors are the largest family of protein tyrosine kinases. The protein-protein interaction between Eph receptors and their protein ligand ephrins and the subsequent bi-directional signaling were recently shown to play an important role in pathological angiogenesis as well as in cancer progression and metastasis. The recent solution of the X-ray crystal structure of EphB2/ephrin-B2 complex made it possible to perform structure-based design of small peptides and peptidomimetics as inhibitors of the protein-protein interaction. Examination of the X-ray crystal structure of the receptor-ligand complex identified a "hot spot" in the receptor-ligand interface, a GHL loop consisting of six amino acids protruding deep into a channel on the Eph receptor surface. Preliminary studies with simple disulfide-linked cyclic peptides indicate that short conformationally restricted cyclic peptides containing the loop residues could bind to the EphB2 receptor with submicromolar affinity. New cyclic peptide analogues and new peptidomimetics were designed in this proposal to test the following two hypotheses: i) better conformational restriction mimicking more accurately the conformation of the G-HL loop as bound to the Eph receptor might offer even better affinity, and ii) molecules based on the distinct conserved sequences of two subclass ephrins might lead to the discovery of subclass-specific small molecule antagonists (possibly also agonists). Two of the conformationally restricted cyclic peptide analogues were already synthesized in our laboratory and are being evaluated by our collaborators. The specific aims of this project are: 1) to determine the receptor binding affinity towards EphB2 and EphB1 receptors of the conformationally restricted cyclic peptides already synthesized, relative to EphA2 and EphA3 receptors, and to modify residues in the conformationally restricted cyclic peptides to those commonly found in ephrin-As in the hope of achieving EphA-specific small molecule antagonists; 2) to synthesize and evaluate peptidomimetics using D-glucose as a scaffold incorporating groups believed to be critical in binding to EphB receptors; and 3) to synthesize and evaluate a hybrid structure containing the D-glucose scaffold and a cyclic tripeptide in efforts to further restrict the conformation and increase the affinity towards EphB receptors. Successful antagonists can be used as pharmacological probes for the elucidation of ephdn and Eph functions in various physiological and pathological processes and as potential antiangiogenic and anticancer agents for the treatment of cancer.